FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule

被引:165
作者
Truong, K
Sawano, A
Mizuno, H
Hama, H
Tong, KI
Mal, TK
Miyawaki, A
Ikura, M [1 ]
机构
[1] Univ Toronto, Ontario Canc Inst, Div Mol & Struct Biol, Toronto, ON M5G 2M9, Canada
[2] Univ Toronto, Dept Med Biophys, Toronto, ON M5G 2M9, Canada
[3] RIKEN, Inst Phys & Chem Res, Brain Sci Inst, Adv Technol Dev Ctr,Lab Cell Funct & Dynam, Wako, Saitama 3510198, Japan
[4] Brain Sci & Life Technol Res Fdn, Brain Sci Res Div, Tokyo 1750094, Japan
关键词
D O I
10.1038/nsb728
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Intracellular Ca2+ acts as a second messenger that regulates numerous physiological cellular phenomena including development, differentiation and apoptosis. Cameleons, a class of fluorescent indicators for Ca2+ based on green fluorescent proteins (GFPs) and calmodulin (CaM), have proven to be a useful tool in measuring free Ca2+ concentrations in living cells. Traditional cameleons, however, have a small dynamic range of fluorescence resonance energy transfer (FRET), making subtle changes in Ca2+ concentrations difficult to detect and study in some cells and organelles. Using the NMR structure of CaM bound to the CaM binding peptide derived from CaM-dependent kinase kinase (CKKp), we have rationally designed a new cameleon that displays a twofold increase in the FRET dynamic range within the physiologically significant range of cytoplasmic Ca2+ concentration of 0.05-1 muM.
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页码:1069 / 1073
页数:5
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